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研究生：	劉振發 Liou, Jenn-Fa
論文名稱：	利用禽蛋生產具功能性的抗體(IgY) Production of functional antibody (IgY) in poultry eggs
指導教授：	陳立人 Chen, Lih-Ren 張敏政 Chang, Ming-Chung
學位類別：	博士 Doctor
系所名稱：	生物科學與科技學院 - 生物科技研究所 Institute of Biotechnology
論文出版年：	2012
畢業學年度：	100
語文別：	英文
論文頁數：	90
中文關鍵詞：	雞、免疫、雞蛋、蛋黃免疫球蛋白、抗71型腸病毒IgY 、抗大腸桿菌IgY 、抗脂肪分解酶IgY
外文關鍵詞：	Chicken, immunization, egg, Immunoglobulin in yolk (IgY), anti-EV71 IgY, anti-E. coli IgY, anti-lipase IgY
相關次數：	點閱：105 下載：1
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近年來抗體被廣泛應用於人類和家畜禽的被動免疫治療，利用其和抗原專一性敖合(chelating)方式去結合專一的病原菌，阻止其和宿主細胞接觸進而發生感染。這種方式不會誘發病原突變而產生抗藥性，抗體也不會攻擊宿主體內其他細胞或細菌造成傷害。利用禽蛋生產IgY抗體漸受重視，在多數研究皆可獲得抗體，減輕病原之影響，證明利用口服專一性IgY抗體方式，具有預防和治療疾病潛能。
本論文共分三部分來探討蛋黃免疫球蛋白抗體，在用於口服被動免疫治療的評估。分別為利用感染人類的71型腸病毒株、引起初生仔豬早發性下痢的大腸桿菌株及脂肪分解酶(Lipase)為抗原，來對產蛋母雞進行免疫注射，利用母雞分別來生產具有抗人類71型腸病毒(anti-EV71 IgY)、抗引起初生仔豬早發性下痢的大腸桿菌(anti-E. coli IgY)與抗脂肪分解酶(anti-lipase IgY)之具特殊功能性的蛋黃免疫球蛋白抗體(IgY)。結果一：在生產anti-EV71 IgY 部分，經以小鼠的動物模式實驗結果顯示，anti-EV71 IgY不論是口服或腹腔注射給予，均讓小鼠對於人類71型腸病毒有明顯的防治效果。結果二：在anti-E. coli IgY ，在抑菌及誘發仔豬下痢防治功效的試驗中，顯示專一性 anti-E. coli IgY抗體在63 μg/mL WSF，具有抑制大腸桿菌生長；在誘發仔豬下痢防治功效的試驗結果，顯示21日齡離乳仔豬餵食 anti-E. coli IgY對感染大腸桿菌所造成早發性下痢的疾病有防治的功效。結果三：在anti-lipase IgY 部分在利用小鼠為動物模式測試證實anti-lipase IgY具有生物活性，能減少脂肪堆積，降低小鼠體重增加，具有控制體重的功效。
關鍵字:雞、免疫、雞蛋、蛋黃免疫球蛋白、抗71型腸病毒IgY、抗大腸桿菌IgY、抗脂肪分解酶IgY。

Passive immunization by specific antibodies has been studied extensively against a lot of pathogens in both humans and animals. The increase in antibiotic-resistant bacteria and the demand to treat pathogens that do not respond to antibiotics, such as viral pathogens, has much research into antibodies as an alternative to antibiotics. The potential of orally administered immunoglobulin in yolk (IgY) for the prevention and treatment of a lot of pathogens have been studied for many years.
This thesis was including three parts. The part Ι were to investigate the potential of IgY for oral immunotherapy with focus was conducted to characterize the specific IgY against human enterovirus type 71 (EV-71). The results of the neutralization effect of specific IgY in EV71-challenged mice demonstrate that the EV71-specific IgY, either by intraperitoneal injection or oral administration, was able to significantly reduce the morbidity and mortality in EV71 infected mice pups. Part II: were to investigate the immunization response of breeds of chickens to Escherichia coli (E. Coli) antigen and the function of specific IgY produced. The results of this specific IgY can inhibit the growth of activated E. coli at a concentration above 63 μg/mL WSF. The chicken egg-yolk antibodies in this study were able to prevent the diarrhea experimentally induced by ETEC in 21-day-old weaned piglets. Part III : Production of body weight control antibody in poultry eggs. The results of body weight control efficiency of specific IgY (anti-lipase IgY) determined using murine model indicated that this anti-lipase IgY was able to prevent obesity induced by a high-fat diet in mice.
Key word: Chicken, Immunization, egg, Immunoglobulin in yolk (IgY), anti-EV71 IgY, anti-E. coli IgY, anti-lipase IgY.

中文摘要...................................................I
Abstract.................................................II
誌謝.....................................................III
Abbreviations.............................................IV
Table of Contents........................................VI 
List of Figures...........................................X
List of Tables...........................................XII
Preface...................................................1 

Chapter 1
Introduction..............................................3
1.Immunoglobulin in yolk (IgY)............................3
1-1 Chicken immunoglobulins...............................3
1-2 Transfer of IgY from hen to egg.......................4
1-3 Structure and Characteristics of Avian IgY Versus  
    Mammal IgG............................................4
2.IgY Application.........................................6
2-1 Advantages of Chicken IgY Antibodies..................6
2-2 IgY in immunological assays and diagnostics ..........7
2-3 IgY for oral immunotherapy............................8
2-4 Oral delivery of IgY..................................8

Chapter 2
Aim......................................................10
Part I : Produce of IgY against human Enterovirus 71
         (anti-EV71 IgY).................................10 
Part II : Produce of IgY against E. coli infection 
          in pigs........................................10
Part III : Production of body weight control antibody 
          in poultry eggs................................10

Chapter 3
Production of IgY against human Enterovirus 71(anti-EV71 IgY)
1.Introduction...........................................12
2.Materials and methods..................................13
2.1.Virus strains and cells..............................13
2.2. Immunization of Chickens............................14
2.3. Isolation and purification of IgY...................14 
2.4. Enzyme-Linked ImmunoSorbent Assay (ELISA)...........15
2.5. Western Blot Analysis...............................16
2.6. TCID50 and EV71 neutralization tests................16
2.7. EV71 infection and IgY protection tests.............17
3.  Results
3.1. Abstract............................................18
3.2. Production of IgY against EV71......................18
3.3. The neutralization titer determination of 
     specific IgY .......................................21
3.4. Neutralization effect of specific IgY in EV71 
     challenged mice.....................................23
4.Discussion.............................................27
5.Conclusion.............................................33

Chapter 4
Produce of IgY against E. coli infection in pigs
1.Introduction...........................................34
2.Materials and methods..................................34
2.1. Preparation of E. coli antigens.....................34
2.2. Immunization of Chickens............................35
2.3. Isolation and Purification of IgY...................36
2.4. Antibody Activity Determination for Serum 
     IgG and total IgY specific Anti-E. coli IgY.........36
2.5. Determination of In Vitro Inhibitory efficiency
     for anti- E. coli IgY17.............................37
2.6. Immunofluorescence microscopy analyses..............38
2.7. Passive protection of piglets.......................38
2.8. Statistical.........................................39
3.Results................................................40
3.1. Abstract............................................40
3.2. Production of IgY against E. coli...................40
3.3. In Vitro Inhibitory efficiency of anti-E.coli IgY...48
3.4. Passive protection ofpiglets .......................52
4.Discussion.............................................53
5.Conclusion.............................................57

Chapter 5
Production of body weight control antibody in poultry eggs
1.Introduction...........................................58
2.Materials and methods..................................59
2.1. Immunization of chickens............................59
2.2. Isolation and purification of IgY...................59
2.3. Enzyme-Linked ImmunoSorbent Assay (ELISA )..........60
2.4. Mice................................................61
2.5. Estimation of body and perirenal adipose 
     tissue weights in mice fed a high-fat diet
     for 6 weeks.........................................61
2.6. Serum biochemical parameter analysis................61
2.7. Statistical analysis................................62
3.Results................................................63
3.1. Abstract............................................63
3.2. Production of IgY against lipase....................63
3.3. Body weight and feed efficiency.....................65
3.4. Effects of anti-lipase IgY on the diameter of  
     adipocyte and the weight of the perirenal 
     adipose tissue in mice fed with a high-fat 
     diet................................................68
3.5. Effects of anti-lipase IgY on blood 
     biochemical parameters in mice fed on 
     different diets.....................................70
4.Discussion.............................................71
5.Conclusion.............................................74

References...............................................75
Publication..............................................89
Conferences..............................................90

List of Figures

Figure 1 Structure of IgG and IgY........................5
Figure 2 The content of EV71 specific IgY per egg yolk  
         during the immunization period..................20
Figure 3 The neutralization titer of specific anti-EV71 IgY 
         was determined by the cytopathic effect (CPE) of 
         EV71 on RD cells................................21
Figure 4 Western blot analysis of anti-EV71 IgY..........22
Figure 5 ICR mice showed both limb paralysis and abnormal 
         hair growth after EV71challenge in the IP model..23
Figure 6 The concentration of total IgY content for White  
         Leghorn chickens egg yolk 0-20 weeks post 
         immunization with CM and AF antigen.............43
Figure 7 The concentration of total IgY content for Lohmann 
         chickens egg yolk 0-20 weeks post immunization 
         with CM and AF antigen..........................44
Figure 8 The serum specific IgY concentration of White 
         Leghorn and Lohmann chickens 0-20 weeks after 
         immunization with CM and AF antigen.............45
Figure 9 The production of anti-E. coli IgY in White 
         Leghorn and Lohmann chickens immunized with CM 
         and AF antigen..................................46
Figure 10 The average anti-E.coli IgY content in eggs 
          collected from White Leghorn and Lohmann browns 
          chickens 4-20 weeks after immunization with CM 
          and AF antigen.................................47 
Figure 11 Immunofluorescence micrographs of E. coli K99 
          incubated with specific IgY and non-specific 
          IgY............................................51
Figure 12 The content of total IgY per egg yolk during the 
          immunization period............................64
Figure 13 The content of lipase specific IgY per egg yolk 
          during the immunization period.................65
Figure 14 The changes in body weight of the groups during 
          the experiment.................................66
Figure 15 The diameters of perirenal adipocytes under light 
          microscopy.....................................69 

List of Tables

Table 1 Comparison of avian IgY and mammalian IgG……………………………...5
Table 2 Comparison of rabbit and chicken polyclonal antibody yield during a
 two-week period following the second immunization……………..…….....7
Table 3 Purity of IgY after different procedure of purification……………………20
Table 4 Neutralization titers of EV71 specific IgY from different chickens….........22
Table 5 Effects of IP challenge on EV71a and IgYb treatment………………....26
Table 6. Effects of oral challenge on EV71a and IgYb treatment………….…..26
Table 7 The E. coli inhibitory effects of the anti-E. coli IgY produced from White Leghorn and Lohmann chickens in the 9th week after immunization with
 antigen of commercial CM and AF………………………….………….49
Table 8 The E. coli inhibitory effects of the anti-E. coli IgY produced from White Leghorn and Lohmann chickens in the 13th week after immunization 
with antigen of CM and AF………………………………………….….....50
Table 9 Clinical response of 21-day-old pigs after challenge with E. coli K99 
and treatment with egg-yolk antibodies………………………………..…...52
Table 10 Body weight of mice fed with different diets…………………………….67
Table 11 The diameter of adipocyte and the weight of the perirenal adipose 
tissue in mice fed with different diets………………………………….…..69
Table 12 Effect of different diets on serum biochemical parameters in mice………70

                                    

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